Gemcitabine Immunoassay

ABSTRACT

The present invention comprises novel conjugates and immunogens derived from gemcitabine and unique antibodies generated by using gemcitabine linked immunogens, which conjugates immunogens and antibodies, are useful in immunoassays for the quantification and monitoring of gemcitabine in biological fluids.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part Application of U.S.application Ser. No. 13/114,218 filed May 24, 2011.

FIELD OF THE INVENTION

This invention relates to the field of immunoassays for determining thepresence or quantifying the amount of gemcitabine in human biologicalsamples in order to rapidly determine optimal drug concentrations duringchemotherapy.

BACKGROUND OF THE INVENTION

Cancer is a term used to describe a group of malignancies that all sharethe common trait of developing when cells in a part of the body begin togrow out of control. Most cancers form as tumors, but can also manifestin the blood and circulate through other tissues where they grow. Cancermalignancies are most commonly treated with a combination of surgery,chemotherapy, and/or radiation therapy. The type of treatment used totreat a specific cancer depends upon several factors including the typeof cancer malignancy and the stage during which it was diagnosed.

Gemcitabine is a commonly used cytotoxic agent that is used for thetreatment of Pancreatic Cancer; Poplin et al J Clin Oncol, 27, 23,3778-85, 2009 and Non-Small Cell Lung Cancer; Zinner, R G, et al., Int JRadiat Oncol Biol Phys, 73, 1, 119-27 2009; and Treat, J A, et al., AnnOncol, 2009. Gemcitabine is also used as an adjuvant treatment inpancreatic cancer (Saif, M W, JOP, 10, 4, 373-7 2009; Li, J and M WSaif, JOP, 10, 4, 361-5 2009). Although it is widely used, this compoundhas been associated with debilitating side effects such asmyelosupression, along with liver and kidney damage. By monitoring thelevels of gemcitabine in the body and adjusting the dose these sideeffects can be better controlled and limited in patients.

Gemcitabine is the hydrochloride salt of the formula:

There is often high variable relationship between the dose ofgemcitabine and the resulting serum drug concentration that affectstherapeutic effect. This is especially prevalent in women and elderlypatients. These groups display a lower clearance, resulting in higherplasma concentrations for any given dose. Gemcitabine (I) is metabolizedin the body by cytidine deaminase (CDA) to its main pharmaceuticallyinactive metabolite: 2′,2°-difluoro-2′-deoxyuridine (dFdU) which has theformula:

There are at most only trace amounts of other gemcitabine metabolites inthe blood, plasma or serum of patients. In preparing human biologicalsamples such as blood and plasma samples for immunoassays it isnecessary to use tetrahydrouridine (THU). This preservative acts toinhibit cytidine deaminase activity during the collection of patientsamples to prevent further metabolism of gemcitabine to the inactivemetabolite of the compound of formula II. The preservativetetrahydrouridine has the following formula:

The degree of intra- and inter-individual pharmacokinetic variability ofgemcitabine varies greatly and is impacted by many factors, including:

-   -   Organ function    -   Genetic regulation    -   Disease state    -   Age    -   Time of sampling,    -   Mode of drug administration, and    -   Technique-related administration.

As a result of this variability, equal doses of the same drug indifferent individuals can result in dramatically different clinicaloutcomes, as illustrated below (Hon, Y Y and W E Evans, Clin Chem, 44,2, 388-400 1998.). The effectiveness of the same gemcitabine dosagevaries significantly based upon individual drug metabolism and theultimate serum drug concentration in the patient. Therapeutic drugmanagement would provide the clinician with insight on patient variationin both oral and intravenous drug administrations. With therapeutic drugmanagement, drug dosages could be individualized to the patient, and thechances of effectively treating the cancer without the unwanted sideeffects would be much higher (Nieto, Y, Curr Drug Metab, 2, 1, 53-662001).

In addition, therapeutic drug management of gemcitabine would serve asan excellent tool to ensure compliance in administering chemotherapywith the actual prescribed dosage and achievement of the effective serumconcentration levels. It has been found that variability in serumconcentration is not only due to physiological factors, but can alsoresult from variation in administration technique (Caffo, O, S Fallani,E Marangon, S Nobili, M I Cassetta, V Murgia, F Sala, A Novelli, E Mini,M Zucchetti and E Galligioni, Cancer Chemother Pharmacol, 2010).

Routine therapeutic drug management of gemcitabine would require theavailability of simple automated tests adaptable to general laboratoryequipment. Tests that best fit these criteria are immunoassays such as aradioimmunoassay and an enzyme-linked immunosorbent assay. However thecorresponding antibodies used in these immunoassays must demonstrate abroad cross-reactivity to gemcitabine, without any substantial activityto non-pharmaceutically active gemcitabine metabolites and thepreservative of formula III. In order to be effective in monitoring druglevels of gemcitabine, the antibody should be most specific to theactive compound, gemcitabine and display very low cross-reactivity to nocross-reactivity to the non-pharmaceutically active metabolite,2′,2′-difluoro-2′-deoxyuridine (the compound of Formula II) and thepreservative tetrahydrouridine (the compound of Formula III).

SUMMARY OF INVENTION

In accordance with this invention, a new class of antibodies have beenproduced which are substantially selectively reactive to gemcitabine soas to bind to gemcitabine without any substantial cross reactivity toits major non-pharmaceutically active gemcitabine metabolite,2′,2′-difluoro-2′-deoxyuridine. In addition these antibodies do notreact with the gemcitabine preservative, tetrahydrouridine, which isnecessary in collecting patient samples to stabilize the gemcitabine inthe collected patient samples. By selectively reactive, it is meant thatthese antibodies only react with the pharmaceutically active gemcitabinemolecule and do not substantially react or cross react with thenon-pharmaceutically active gemcitabine metabolites, the most importantand basic blocking metabolite being 2′,2′-difluoro-2′-deoxyuridine andthe preservative, tetrahydrouridine.

It has been found that by using immunogens which are conjugates of animmunogenic carrier having a reactive thiol or amino functional groupwith 5-substituted gemcitabine compounds of the formula:

-   -   wherein B is —CH₂— or

-   -   Y is an organic spacing group;    -   X is a functional group capable of binding to said carrier        through said amino or thiol group; and    -   p is an integer from 0 to 1        or salts thereof; produce antibodies which are specific for        gemcitabine and do not substantially react with or bind with the        non-pharmaceutical active metabolite,        2′,2′-difluoro-2°-deoxyuridine as well as tetrahydrouridine. The        provision of these antibodies which substantially selectively        react with gemcitabine and do not cross react with        2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine allows one        to produce an immunoassay which can specifically detect and        monitor gemcitabine in the fluid samples of patients being        treated with gemcitabine. Also included within this invention        are reagents and kits for said immunoassay.

DETAILED DESCRIPTION

In accordance with this invention, a new class of antibodies is providedwhich are substantially selectively reactive with gemcitabine and do notsubstantially react or cross react with pharmaceutically inactivegemcitabine metabolites, particularly 2°,2′-difluoro-2′-deoxyuridine andthe preservative, tetrahydrouridine. It has been discovered that throughthe use of these derivatives of the compound of Formula IV or saltsthereof, as immunogens, this new class of antibodies of this inventionare provided. It is through the use of these antibodies that animmunoassay, including reagents and kits for such immunoassay fordetecting and/or quantifying gemcitabine in blood, plasma or other bodyfluid samples has been developed. By use of this immunoassay, thepresence and amount of gemcitabine in body fluid samples, preferably ablood or plasma sample, can be detected and/or quantified. In thismanner, a patient being treated with gemcitabine can be monitored duringtherapy and his treatment adjusted in accordance with said monitoring.By means of this invention one achieves the therapeutic drug managementof gemcitabine in cancer patients being treated with gemcitabine as achemotherapeutic agent.

The reagents utilized in the assay of this invention are conjugates of acarrier containing a reactive thiol or amino group with the compounds ofFormula IV or salts thereof. Preferably the carriers contain a polyaminepolymer, which contains a reactive thiol or amino group. In preparingthe immunogens, the carriers are immunogenic polymers which preferablycontain a polyamine polymer, having a reactive thiol or amino group.When used in an immunoassay, these conjugates are competitive bindingpartners with the gemcitabine present in the sample for the binding withthe antibodies of this invention. Therefore, the amount of conjugatereagent which binds to the antibody will be inversely proportional tothe amount of gemcitabine in the sample. In accordance with thisinvention, the assay utilizes any conventional measuring means fordetecting and measuring the amount of said conjugate which is bound orunbound to the antibody. Through the use of said means, the amount ofthe bound or unbound conjugate can be determined. Generally, the amountof gemcitabine in a sample is determined by correlating the measuredamount of the bound or unbound conjugate produced by the gemcitabine inthe sample with values of the bound or unbound conjugate determined froma standard or calibration curve obtained from samples containing knownamounts of gemcitabine, which known amounts are in the range expectedfor the sample to be tested. These studies for producing calibrationcurves are determined using the same immunoassay procedure as used forthe sample.

The conjugates, which include the immunogens, are prepared fromcompounds of the formula IV or salts thereof. The carriers, includingthe immunogens, having a reactive terminal amino or thiol group, arelinked to the ligand portion which has the formula:

-   -   wherein X′ is —CH₂— or a functional linking group, Y, B and p        are as above. This ligand portion may be linked to one or more        active thiol or amino sites on the carrier containing the        polyamine polymer. Preferably these carriers contain a polymer,        most preferred a polyamine polymer, containing a reactive thiol        or amino group.

Definitions

Throughout this description the following definitions are to beunderstood:

The term gemcitabine includes gemcitabine as well as thepharmaceutically acceptable salts of gemcitabine.

The terms “immunogen” and “immunogenic” refer to substances capable ofeliciting, producing, or generating an immune response in an organism.

The term “conjugate” refers to any substance formed from the joiningtogether of two parts. Representative conjugates in accordance with thepresent invention include those formed by the joining together of asmall molecule, such as the compound of formula IV and a large molecule,such as a carrier or a polyamine polymer, particularly protein. In theconjugate the small molecule maybe joined at one or more active sites onthe large molecule. The term conjugate includes the term immunogen.

“Haptens” are partial or incomplete antigens. They are carrier-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling a hapten to a highmolecular weight immunogenic carrier and then injecting this coupledproduct, i.e., immunogen, into a human or animal subject. The hapten ofthis invention is gemcitabine.

As used herein, a “spacing group” or “spacer” refers to a portion of achemical structure which connects two or more substructures such ashaptens, carriers, immunogens, labels, or tracers through a CH₂ orfunctional linking group. These spacer groups will be enumeratedhereinafter in this application. The atoms of a spacing group and theatoms of a chain within the spacing group are themselves connected bychemical bonds. Among the preferred spacers are straight or branched,saturated or unsaturated, carbon chains. Theses carbon chains may alsoinclude one or more heteroatoms within the chain or at termini of thechains. By “heteroatoms” is meant atoms other than carbon which arechosen from the group consisting of oxygen, nitrogen and sulfur. Spacinggroups may also include cyclic or aromatic groups as part of the chainor as a substitution on one of the atoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. A functional linking group may be used to activate, e.g.,provide an available functional site on, a hapten or spacing group forsynthesizing a conjugate of a hapten with a label or carrier orpolyamine polymer.

An “immunogenic carrier,” as the terms are used herein, is animmunogenic substance, commonly a protein or a protein modified to carrya reactive thiol or amino group, that can join with a hapten, in thiscase gemcitabine, thereby enabling these hapten derivatives to induce animmune response and elicit the production of antibodies that can bindspecifically with these haptens. The immunogenic carriers and thelinking groups will be enumerated hereinafter in this application. Amongthe immunogenic carrier substances are included proteins, glycoproteins,complex polyamino-polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host. The polyamino-polysaccharides may be prepared frompolysaccharides using any of the conventional means known for thispreparation.

Also various protein types may be employed as a poly(amino acid)immunogenic carrier. These types include albumins, serum proteins,lipoproteins, etc. Illustrative proteins include bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovinethyroglobulin (BTG) etc. Alternatively, synthetic poly(amino acids) maybe utilized. Alternatively these proteins can be modified so as tocontain a reactive thiol group.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymer built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide may also contain polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

“Poly(amino acid)” or “polypeptide” is a polyamide formed from aminoacids. Poly(amino acids) will generally range from about 2,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000 and usually not more than about 600,000 daltons. There willusually be different ranges, depending on whether an immunogenic carrieror an enzyme is involved.

A “peptide” is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of α-amino acids inwhich the α-amino group of each amino acid residue (except the NH₂terminus) is linked to the α-carboxyl group of the next residue in alinear chain. The terms peptide, polypeptide and poly(amino acid) areused synonymously herein to refer to this class of compounds withoutrestriction as to size. The largest members of this class are referredto as proteins. These polymer peptides can be modified by conventionalmeans to convert the reactive NH₂ terminal group into a terminal SHgroup.

A “label,” “detector molecule,” or “tracer” is any molecule whichproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, immunogen, antibody, or to anothermolecule such as a receptor or a molecule that can bind to a receptorsuch as a ligand, particularly a hapten. Non-limiting examples of labelsinclude radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, or sensitizers; a non-magnetic ormagnetic particle, a solid support, a liposome, a ligand, or a receptor.

The term “antibody” refers to a specific protein binding partner for anantigen and is any substance, or group of substances, which has aspecific binding affinity for an antigen to the exclusion of othersubstances. The generic term antibody subsumes polyclonal antibodies,monoclonal antibodies and antibody fragments.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “carrier” refers to solid particles and/or polymeric polymerssuch as immunogenic polymers such as those mentioned above. Where thecarrier is a solid particle, the solid particle may be bound, coatedwith or otherwise attached to a polyamine polymer to provide one or morereactive sites for bonding to the functional group X in the compounds ofthe formula IV.

The term “reagent kit,” or “test kit,” refers to an assembly ofmaterials that are used in performing an assay. The reagents can beprovided in packaged combination in the same or in separate containers,depending on their cross-reactivity and stability, and in liquid or inlyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. A reagent kit embodying features of the presentinvention comprises antibodies specific for gemcitabine. The kit mayfurther comprise ligands of the analyte and calibration and controlmaterials. The reagents may remain in liquid form or may be lyophilized.

The phrase “calibration and control materials” refers to any standard orreference material containing a known amount of a drug to be measured.The concentration of drug is calculated by comparing the resultsobtained for the unknown specimen with the results obtained for thestandard. This is commonly done by constructing a calibration curve.

The term “biological sample” includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, horses, and other animals. Such substancesinclude, but are not limited to, blood, serum, plasma, urine, cells,organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, and skin.

Reagents and Immunogens

In constructing an immunoassay, a conjugate of gemcitabine isconstructed to compete with the gemcitabine in the sample for bindingsites on the antibodies. In the immunoassay of this invention, thereagents are the conjugates of the 5′ substituted gemcitabinederivatives of the compounds of formula IV and the antibodies having theaforementioned requisite properties. In the compounds of formula IV-B,the linker spacer constitutes the —B—(Y)_(p)—X′ portion of thismolecule. In these linkers X′ and the spacer —B—(Y)_(p)—X′ areconventional in preparing conjugates and immunogens. Any of theconventional spacer-linking groups utilized to prepare conjugates andimmunogens for immunoassays can be utilized in the compounds of formulaIV-B. Such conventional linkers and spacers are disclosed in U.S. Pat.No. 5,501,987 and U.S. Pat. No. 5,101,015.

Among the preferred spacer groups are included the spacer groupshereinbefore mentioned. Particularly preferred spacing groups are groupssuch as alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6 with alkylene being the especially preferred spacing group. Withrespect to the above structures of the spacing group designated by Y,the functional group X is connected at the terminal position at theright side of the structure i.e. where (CH₂)_(m) and (CH₂)_(o) arelocated.

In the compounds of formula IV-B, X′ is —CH₂— or a functional grouplinking the spacer, to an amine or thiol group on the polymeric carrier.The group X′ is the result of the terminal functional group X in thecompounds of Formula IV which is capable of binding to the amino orthiol group in the polyamine polymer used as either the carrier or asthe immunogen. Any terminal functional group capable of reacting with anamine or thiol group can be utilized as the functional group X in thecompounds of formula IV. These terminal functional groups preferablyincluded within X are:

—N═C═R_(4′) or

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur. The —N═c═R_(4′)radical can be an isocyanate or as isothiocyanate. The active estersformed by —OR₃ include imidoester, such as N-hydroxysuccinamide,i-hydroxy benzotriazole and p-nitrophenyl ester. However any activeester which can react with an amine or thiol group can be used.

The carboxylic group and the active esters are coupled to the carrier orimmunogenic polymer by conventional means. The amine group on thepolyamine polymer, such as a protein, produces an amide group whichconnects the spacer to the polymeric immunogens or carrier to form theconjugates of this invention.

When X in the compound of formula IV is

—N═C═R_(4′) or

these compounds preferably react with the free amino group of thepolymeric or immunogenic carrier.

On the other hand, when X in the compound of formula IV is the maleimideradical of the formula

this compound preferably reacts with the thiol (or SH) group which maybe present on the polymeric or protein carrier, including theimmunogens. In this case where X is the maleimide radical the compoundof the formula IV has the structure:

In accordance with a preferred embodiment, these compounds of formulaIV-C are reacted to attach to a polymeric protein which has beenmodified to convert an amino group to a thiol group. This can be done bythe reacting a free amino group of a polymeric protein carrier with acompound of the formula

wherein R₁₅ is a thiol protecting group;

R₃ is as above; and

v is an integer of from 1 to 4.

This reaction is carried out in an aqueous medium by mixing the proteincontaining carrier with the compound of formula V in an aqueous medium.In this reaction temperature and pressure are not critical and thereaction can be carried out at room temperature and atmosphericpressure. Temperatures of from 10° C. to 25° C. are generally preferred.In the compound of formula V which is reacted with the compound ofFormula IV-C, any conventional thiol protecting agent can be utilized.The thiol protecting groups are well known in the art with2-pyridyldthio being the preferred protecting group. By this reaction,the thiol group, SH— becomes the functional group of the carrier whichbonds the compound of formula IV to the remainder of the carrier

In the next step, before reacting with the compound of Formula IV-C withthe thiol modified carrier, the thiol protecting group of carrier isremoved by conventional means from the resulting reaction product whichis formed by reacting the compound of formula V with the carrier. Anyconventional means for removing a thiol protecting group can be utilizedin carrying out this reaction. However, in utilizing a means to removethe thiol protecting group, care must be taken that the reactants besoluble in the aqueous medium and do not in any way destroy or harm thepolyamine polymer contained in the carrier. A preferred means forremoving this protecting group is by the use of dithiothreitol as anagent to reduce the resultant condensation product. This reduction canbe carried out by simply adding the reducing agent to the reactionmedium without utilizing higher pressures or temperatures. Thisreduction can be carried out at room temperature and atmosphericpressure.

While the above method represents one means for converting a reactiveterminal amino group on the polyamine polymeric containing carrier to athiol group, any conventional means for carrying out this conversion canbe utilized. Methods for converting terminal amino groups on polyaminepolymeric containing carriers to thiol groups are well known in the artand can be employed in accordance with this invention.

The reaction of the polymeric polyamine containing carrier having aterminal reactive thiol group with the compound of formula IV where X isa functional group capable of binding to the terminal thiol groupcarried by the carrier can be carried out by conventional means. Themaleimide of IV C is reacted with the thiol group carried by thepolyamine polymeric carrier. Any well known means for addition of athiol across a maleimide double bond can be utilized in producing theconjugates of formula IV which are conjugated through a thiol bridge.

In the conjugates, bonded through amide bonds which conjugates includethe immunogens of the present invention, the chemical bond between thecarboxyl group containing gemcitabine haptens and the amino groups onthe carrier or immunogen can be obtained using a variety of methodsknown to one skilled in the art. It is frequently preferable to formamide bonds by first activating the carboxylic acid moiety of thegemcitabine hapten in the compound of formula IV or theirpharmaceutically acceptable salts by reacting the carboxy group with aleaving group reagent (e.g., N-hydroxysuccinimide,i-hydroxybenzotriazole, p-nitrophenol and the like). An activatingreagent such as dicyclohexylcarbodiimide, diisopropylcarbodiimide andthe like can be used. The activated form of the carboxyl group in thegemcitabine hapten of the compound of Formula IV or its pharmaceuticallyacceptable salts is then reacted in a buffered solution containing theprotein carrier.

In preparing the amino bonded conjugates where the gemcitabinederivative of formula IV contains a primary or secondary amino group aswell as the carboxyl group, it is necessary to use an amine protectinggroup during the activation and coupling reactions to prevent theconjugates from reacting with themselves. Typically, the amines on thegemcitabine derivative of formula IV are protected by forming thecorresponding N-trifluoroacetamide, N-tertbutyloxycarbonyl urethane(N-t-BOC urethane), N-carbobenzyloxy urethane or similar structure. Oncethe coupling reaction to the immunogenic polymer or carrier has beenaccomplished, as described above, the amine protecting group can beremoved using reagents that do not otherwise alter the structure of theimmunogen or conjugate. Such reagents and methods are known to oneskilled in the art and include weak or strong aqueous or anhydrousacids, weak or strong aqueous or anhydrous bases, hydride-containingreagents such as sodium borohydride or sodium cyanoborohydride andcatalytic hydrogenation. Various methods of conjugating haptens andcarriers are also disclosed in U.S. Pat. No. 3,996,344 and U.S. Pat. No.4,016,146, which are herein incorporated by reference.

On the other hand in preparing amino conjugates where X is a terminalisocyanate or thioisocyanate radical in the compound of formula IV,these radicals when reacted with the free amine of a polyamine polymerproduce the conjugate or immunogen of formula IV-B where X′ is NH

where R₄ is as above, which functionally connects with the amino groupon the polyamine carrier or on the immunogenic polypeptide.

In preparing the amino conjugates of the compounds of formula IV where Xis an aldehyde group these compounds may be connected to the amine groupof the polyamine polypeptide or carrier through an amine linkage byreductive amination. Any conventional method of condensing an aldehydewith an amine such as through reductive amination can be used to formthis linkage. In this case, X′ in the ligand portion of formula IV-B- is—CH₂.

The compound of formula IV and from this compound, the compound offormula IV-B, are prepared from gemcitabine (the compound of formula I).However in preparing the compound of formula IV, from the compound offormula I, it is necessary to selectively protect the hydroxy group ofthat 3′ position and the amino group at the 4 position on the compoundof formula I, what affecting the free hydroxy group at the 5′ positionto produce a compound of the formula.

-   -   wherein R₁₀ is a hydrolyzable hydroxy protecting group; and R₁₁        is a hydrolyzable amino protecting groups

In preparing the compound of formula I-C the compound of formula I isreacted to convert the free hydroxy group to a hydrolyzable hydroxyprotecting group. Any conventional method of converting a free hydroxygroup into a hydrolyzable hydroxy protecting group can by used. Thisreaction should occur under mild alkaline conditions, so that thehydroxy group at the 3′ position is protected while leaving the hydroxygroup at the 5′ position free. The hydroxy group at the 3′ position inthe compound of formula I-C is far more reactive than the hydroxy groupat the 5′ position. Therefore under mild alkaline aqueous conditionssuch as using sodium bicarbonate in an aqueous medium will provide aprotecting group at the 3′ hydroxy position without affecting thehydroxy group at the 5′ position. Any conventional hydroxy protectinggroup which is easily hydrolyzable can be utilized. The preferredhydroxy protecting group is a tertiary butoxy carbonyl group formed byreacting the compound of formula I with tertiary butoxy carbonate undermildly alkaline aqueous conditions at room temperature. Any otherconventional hydroxy protecting groups can be utilized. Among thepreferred hydroxy protecting groups are the ester groups formed byreacting the 3′ hydroxy group in the compound of formula I with aalkanoic acid under mild alkaline conditions to form the ester at the 3′position while leaving the hydroxy group at the 5′ position free. Thecompound of formula I with the protected 3′ hydroxy group can beconverted to the compound of formula I-C by the same reaction that wasused to protect the hydroxy group at the 3′ position except thatelevated temperatures i.e. from 35° C. to 70° C. are utilized. In thismanner the compound of formula I-C is formed from the compound offormula I.

The 5′-substituted compounds of formula IV where B is —CH₂— are formedby reacting the 5′-hydroxy group of gemcitabine with a halide of theformula:

halo-CH₂—(Y)_(p)—X  VIII-B

-   -   wherein p, Y and X are as above.

In the next step of forming the compound of formula IV from gemcitabine,any conventional means of reacting an alcohol to form ethers can beutilized to condense the compound of formula VIII-B with the 5′ hydroxyposition on the gemcitabine. The use of a halide in the compound offormula VIII-B provides an efficient means for forming such ethers bycondensing with the alcohol. On the other hand, where Y in the compoundof formula VIII-B contains functional groups, which may interfere withthis reaction to form the compound of formula II-B, these functionalgroups can be protected by means of suitable protecting groups which canbe removed after this reaction as described hereinabove.

The 5′-substituted compounds of formula IV where B is

is produced by reacting 5′-hydroxy group on gemcitabine with an aminocompound of the formula:

NH₂—CH₂—(Y)_(p)—X  IX

-   -   wherein X, Y and p are as above.

After first converting the 5′-hydroxy group on gemcitabine to thechloroformatic group

Any conventional means of converting a hydroxy group to a chloroformaticgroup can be used. After the formulation of a chloroformate, the halogroup of the chloroformate is condensed with the amine group in thecompound of formula IX. Prior to this reaction, the reactive group ongemcitabine and/or on the compound of formula IX are protected asdescribed hereinabove with a conventional protecting group. Theseprotecting groups can be removed after this halide condensation byconventional means such as described hereinbefore.

The compound of formula IV-B can be converted into the immunogens and/orthe conjugate reagents of this invention by reacting these compoundswith a polyamine or a polypeptide carrier which contains a terminalamino group. The same polypeptide can be utilized as the carrier and asthe immunogenic polymer carrier in the immunogen of this inventionprovided that the polyamine or polypeptide carrier used to generate theantigen is immunologically active. However, to form the conjugates,these polymers need not produce an immunological response as needed forthe immunogens. In accordance with this invention, the variousfunctional groups represented by X in the compounds of formula IV-B canbe conjugated to the polymeric material by conventional means ofattaching a functional group to an amine or thiol group contained withinthe polymeric carrier.

The compounds of formula IV as either the reagent, conjugate includingthe immunogen prepared therefrom can be present or used in theimmunoassay of this invention in its salt form or as a free base. Thefree amino group in the compound of formula IV and in the conjugateincluding immunogen prepared therefrom readily forms salts with acidspreferably pharmaceutically acceptable acids. Any acid salt of thecompound of formula IV and the conjugates including immunogen preparedtherefrom can be used in this invention. These salts s including bothinorganic and organic acids such as, for example, acetic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like.Particularly preferred are fumaric, hydrochloric, hydrobromic,phosphoric, succinic, sulfuric and methanesulfonic acids.

Antibodies

The present invention also relates to novel antibodies includingmonoclonal antibodies to gemcitabine produced by utilizing theaforementioned immunogens. In accordance with this invention it has beenfound that these antibodies produced in accordance with this inventionare selectively reactive with gemcitabine and do not react withnon-pharmaceutically active metabolites and other compounds which wouldinterfere with immunoassays for gemcitabine. The most problematic ofthese gemcitabine metabolites is 2′,2′-difluoro-2′-deoxyuridine and themost problematic preservative is tetrahydrouridine. The ability of theantibodies of this invention not to react with these inactivemetabolites and this preservative makes these antibodies particularlyvaluable in providing an immunoassay for gemcitabine.

The present invention relates to novel antibodies and monoclonalantibodies to gemcitabine. The antisera of the invention can beconveniently produced by immunizing host animals with the immunogens ofthis invention. Suitable host animals include rodents, such as, forexample, mice, rats, rabbits, guinea pigs and the like, or highermammals such as goats, sheep, horses and the like. Initial doses,bleedings and booster shots can be given according to accepted protocolsfor eliciting immune responses in animals, e.g., in a preferredembodiment mice received an initial dose of 100 ug immunogen/mouse, i.p.and one or more subsequent booster shots of between 50 and 100 ugimmunogen/mouse over a six month period. Through periodic bleeding, theblood samples of the immunized mice were observed to develop antibodiesagainst gemcitabine utilizing conventional immunoassays. These methodsprovide a convenient way to screen for hosts which are producingantisera having the desired activity. The antibodies were also screenedagainst the major pharmaceutically inactive metabolites of gemcitabine,particularly 2′,2′-difluoro-2′-deoxyuridine and the preservative istetrahydrouridine and showed no substantial binding to these compounds

Monoclonal antibodies are produced conveniently by immunizing Balb/cmice according to the above schedule followed by injecting the mice with100 ug immunogen i.p. or i.v. on three successive days starting fourdays prior to the cell fusion. Other protocols well known in theantibody art may of course be utilized as well. The completeimmunization protocol detailed herein provided an optimum protocol forserum antibody response for the antibody to gemcitabine.

Blymphocytes obtained from the spleen, peripheral blood, lymph nodes orother tissue of the host may be used as the monoclonal antibodyproducing cell. Most preferred are B lymphocytes obtained from thespleen. Hybridomas capable of generating the desired monoclonalantibodies of the invention are obtained by fusing such B lymphocyteswith an immortal cell line, which is a cell line that which imparts longterm tissue culture stability on the hybrid cell. In the preferredembodiment of the invention the immortal cell may be a lymphoblastoidcell or a plasmacytoma cell such as a myeloma cell. Murine hybridomaswhich produce gemcitabine monoclonal antibodies are formed by the fusionof mouse myeloma cells and spleen cells from mice immunized againstgemcitabine-protein conjugates. Chimeric and humanized monoclonalantibodies can be produced by cloning the antibody expressing genes fromthe hybridoma cells and employing recombinant DNA methods now well knownin the art to either join the subsequence of the mouse variable regionto human constant regions or to combine human framework regions withcomplementary determining regions (CDR's) from a donor mouse or ratimmunoglobulin. An improved method for carrying out humanization ofmurine monoclonal antibodies which provides antibodies of enhancedaffinities is set forth in International Patent Application WO 92/11018.

Polypeptide fragments comprising only a portion of the primary antibodystructure may be produced, which fragments possess one or moreimmunoglobulin activities. These polypeptide fragments may be producedby proteolytic cleavage of intact antibodies by methods well known inthe art, or by inserting stop codons at the desired locations inexpression vectors containing the antibody genes using site-directedmutageneses to produce Fab fragments or (Fab′)₂ fragments. Single chainantibodies may be produced by joining VL and VH regions with a DNAlinker (see Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883(1988) and Bird et al., Science, 242:423-426 (1988)).

The antibodies of this invention are selective for gemcitabine withouthaving any substantial cross-reactivity or reactivity with the majorpharmaceutically non active metabolite of gemcitabine which is2′,2′-difluoro-2′-deoxyuridine and the gemcitabine preservative istetrahydrouridine. By having no substantial cross-reactivity it is meantthat the antibodies of this invention have a cross reactivity, basedupon their reactivity with gemcitabine with its non-pharmaceuticallyactive metabolite, 2′,2′-difluoro-2′-deoxyuridine and its preservativetetrahydrouridine, of less than 20%. Those antibodies having a crossreactivity of less than 15% are preferred and those antibodies having across reactivity of at most 1% are especially preferred, whichpercentages are based upon the reactivity of these antibodies withgemcitabine.

Immunoassays

In accordance with this invention, the conjugates and the antibodiesgenerated from the immunogens of the compounds of IV or salts thereofcan be utilized as reagents for the determination of gemcitabine inpatient samples. This determination is performed by means of animmunoassay. Any immunoassay in which the reagent conjugates formed fromthe compounds of IV or salts thereof compete with the gemcitabine in thesample for binding sites on the antibodies generated in accordance withthis invention can be utilized to determine the presence of gemcitabinein a patient sample. The manner for conducting such an assay forgemcitabine in a sample suspected of containing gemcitabine, comprisescombining an (a) aqueous medium sample, (b) an antibody to gemcitabinegenerated in accordance with this invention and (c) the conjugatesformed from the compounds of formula IV or salts thereof. The amount ofgemcitabine in the sample can be determined by measuring the inhibitionof the binding to the specific antibody of a known amount of theconjugate added to the mixture of the sample and antibody. The result ofthe inhibition of such binding of the known amount of conjugates by theunknown sample is compared to the results obtained in the same assay byutilizing known standard solutions of gemcitabine.

Various means can be utilized to measure the amount of conjugate formedfrom the compounds of formula IV or salts thereof bound to the antibody.One method is where binding of the conjugates to the antibody causes adecrease in the rate of rotation of a fluorophore conjugate. The amountof decrease in the rate of rotation of a fluorophore conjugate in theliquid mixture can be detected by the fluorescent polarization techniquesuch as disclosed in U.S. Pat. No. 4,269,511 and U.S. Pat. No.4,420,568.

On the other hand, the antibody can be coated or absorbed onnanoparticles so that when these particles react with the gemcitabineconjugates formed from the compounds formula IV or salts thereof, thesenanoparticles form an aggregate. However, when the antibody coated orabsorbed nanoparticles react with the gemcitabine in the sample, thegemcitabine from the sample bound to these nanoparticles does not causeaggregation of the antibody nanoparticles. The amount of aggregation oragglutination can be measured in the assay mixture by absorbance.

On the other hand, these assays can be carried out by having either theantibody or the gemcitabine conjugates attached to a solid support suchas a microtiter plate or any other conventional solid support includingsolid particles. Attaching antibodies and proteins to such solidparticles is well known in the art. Any conventional method can beutilized for carrying out such attachments. In many cases, in order toaid measurement, labels may be placed upon the antibodies, conjugates orsolid particles, such as radioactive labels or enzyme labels, as aids indetecting the amount of the conjugates formed from the compounds offormula IV or salts thereof which is bound or unbound with the antibody.Other suitable labels include chromophores, fluorophores, etc.

As a matter of convenience, assay components of the present inventioncan be provided in a kit, a packaged combination with predeterminedamounts of new reagents employed in assaying for gemcitabine. Thesereagents include the antibody of this invention, as well as, theconjugates formed from the compounds of formula IV or salts thereof. Inaddition to these necessary reagents, additives such as ancillaryreagents may be included in these kits, for example, stabilizers,buffers and the like. The relative amounts of the various reagents mayvary widely to provide for concentrations in solution of the reagentswhich substantially optimize the sensitivity of the assay. Reagents canbe provided in solution or as a dry powder, usually lyophilized,including excipients which on dissolution will provide for a reagentsolution having the appropriate concentrations for performing the assay.

EXAMPLES

In the examples, the following abbreviations are used for designatingthe following:

-   -   EtOAc Ethyl acetate    -   Na₂CO₃ Sodium Bicarbonate    -   Boc₂O Di-tert-butyl dicarbonate    -   CDI 1,1′-carbonyldiimidazole    -   Na₂SO₄ Sodium Sulfate    -   CH₂Cl₂ Dichloromethane    -   THF Tetrahydrofuran    -   N₂ Nitrogen gas    -   THF tetrahydrofuran    -   TFA trifluoroacetic acid    -   DMSO Dimethylsulfoxide    -   s-NHS sulfo-N-hydroxy succinimide    -   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   KLH Keyhole Limpet Hemocyanin    -   BSA Bovine serum albumin    -   PBS Phosphate buffered saline    -   NaCl sodium chloride    -   HRP horse radish-peroxidase    -   ANS 8-Anilino-1-naphthalenesulfonic acid    -   TMB 3,3′,5,5′-Tetramethylbenzidine    -   TRIS Tris(hydroxymethyl)aminomethane hydrochloride    -   di-H₂O deionized water

The phosphate buffer composition has an aqueous solution containing

-   -   15.4 mM Sodium phosphate dibasic (Na₂HPO₄)    -   4.6 mM Sodium phosphate monobasic (NaH₂PO₄)

pH=7.2±0.10

In the Examples, Scheme 1 and Scheme 2 below set forth the specificcompounds prepared and referred to by numbers in the Examples. Theschemes are as follows:

Example 1 Preparation of 5′-O—N-carbonyl(gemcitabine)-6′-aminocaproate[7] (scheme 1)

Compound [1] (1.2 g, 4.0 mmol) and Boc₂O (0.88 g, 4.0 mmol) were stirredin dioxane (60 mL) and a solution of Na₂CO₃ (2.12 g, 20.0 mmol) in water(15 mL) was added. The reaction mixture was stirred at 25° C. for 48hours to produce [2] in the mixture. Water (40 mL) was added to thereaction mixture and the product [2] was extracted with EtOAc. The EtOAcorganic phase was washed with brine, dried (Na₂SO₄), and evaporated to awhite solid, which was then triturated with 10% CH₂Cl₂/hexanes to obtaincompound [2] (1.26 g, 87%).

Compound [2] (1.25 g, 3.44 mmol) and Boc₂O (7.52 g, 34.40 mmol) weremixed in dioxane (100 mL) and heated at 37° C. for 48 hours to provide[3]. The solvent was evaporated to a white solid and the white solid wastriturated with 10% CH₂Cl₂/hexanes to obtain the compound [3] (1.30 g,82%).

Compound [3] (1.30 g, 2.80 mmol) and 1,1′-carbonyldiimidazole (0.52 g,3.20 mmol) were mixed in THF (20 mL) and heated at 50° C. for 6 hours.The solvent was evaporated, the residue was dissolved in EtOAc, washedwith water, dried with Na₂SO₄, and the solvent was evaporated to givecompound [4] as a white solid (1.60 g, 100%).

Compound [4] (1.50 g, 2.69 mmol) and compound [5] (0.60 g, 3.23 mmol)were mixed in THF (20 mL) and heated at 50° C. for 24 hours. Thereaction mixture was diluted with EtOAc, sequentially washed with waterand brine, dried with Na₂SO₄, and evaporated to a white solid. Thismaterial was purified by flash chromatography with 10-50% EtOAc/hexanesto obtain compound [6] (1.40 g, 77%).

Compound [6] (1.40 g, 2.07 mmol) was dissolved in anhydrous CH₂Cl₂ (15mL) and TFA (15 mL) was added to the stirred solution at 0° C. under N₂.The stirring was continued at 0° C. for 3 hours and then at 15° C. for 1hour. The solvent was removed under reduced pressure, and the resultingresidue was dissolved in water and lyophilized to isolate compound [7](1.04 g, 94%) as a white powder.

Example 2 Preparation of5′-O—N-carbonyl-(gemcitabine)-6′-methylcarbamoyl benzoic acid [12](Scheme 2)

Compound [4] (0.60 g, 1.08 mmol) and compound [10] (0.38 g, 1.19 mmol)were mixed in THF (20 mL) and heated at reflux for 24 hours. Thereaction mixture was diluted with EtOAc, washed sequentially with waterand brine, dried with Na₂SO₄, and evaporated to a white solid. Thismaterial was purified by flash chromatography with 10-90% EtOAc/hexanesto obtain compound [11] (0.47 g, 54%).

Compound [11] (0.47 g, 0.58 mmol) was dissolved in anhydrous CH₂Cl₂ mL)and TFA (10 mL) was added at 0° C. under N2. The stirring was continuedat 0° C. for 3 h and then at 15° C. for 1 h. The solvent was removedunder reduced pressure and the resulting residue was dissolved in waterand lyophilized to isolate compound [12] (0.33 g, 85%) as an off-whitepowder.

Example 3 General Method for Preparing s-NHS Activated Drug Derivativesfrom the Corresponding Acids [7] & [12]

In Example 3a and 3b, Gemcitabine acid derivatives [7] & [12] wereactivated with EDC and s-NHS to produce the s-NHS activated esters ofgemcitabine [8] & [13] for eventual conjugation to proteins (Examples 4and 5).

Example 3a Preparation of s-NHS activated ester5′-O—N-carbonyl(gemcitabine)-6′-aminocaproate

Compound [7], Example 1, Scheme 1, (101.3 mg) was dissolved in 10 mL ofDMSO to which was added s-NHS (121.7 mg) and EDC (107.1 mg). Thereaction mixture was stirred for 20 hours at ambient temperature under anitrogen atmosphere to produce compound [8]. The reaction mixture wasused directly in Examples 4 and 5a.

Example 3b Preparation of s-NHS activated ester5′-O—N-carbonyl-(gemcitabine)-6′-methylcarbamoyl benzoic acid [13]

Compound [12], Example 2, Scheme 2 (22.7 mg) was dissolved in 2.2 mL ofDMSO and s-NHS (19.2 mg) and EDC (21.9 mg) were added. The reactionmixture was stirred for 20 hours at ambient temperature under a nitrogenatmosphere to produce compound [13]. The reaction mixture was useddirectly in Example 5b.

Example 4 Preparation of the Gemcitabine-KLH Conjugate [9]

A protein solution of KLH was prepared by dissolving 300 mg of KLH in 15mL of phosphate buffer (50 mM, pH 7.5), followed by addition of 4.74 mLof compound [8] prepared in Example 3a. The reaction mixture of KLH andcompound [8] were allowed to stir for 20 hours at room temperature toproduce the gemcitabine KLH conjugate [9]. The gemcitabine KLH conjugate[9] was then purified by dialysis against 30% DMSO in phosphate buffer(50 mM, pH 7.5) at room temperature. Thereafter the DMSO proportion wasreduced stepwise: 20%, 10% and 0%. The last dialysis was performedagainst phosphate buffer at 4° C. The gemcitabine KLH conjugate [9] wascharacterized by ultraviolet-visible spectroscopy. The conjugate wasdiluted to a final concentration of 2 mg/mL in phosphate buffer (50 mM,pH 7.5).

Example 5a Preparation of BSA Conjugate [9] with Activated Hapten,Gemcitabine [8]

A protein solution of BSA was prepared by dissolving 1 g BSA inphosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL.To this protein solution was added 0.83 mL of s-NHS activatedgemcitabine derivative [8] prepared in Example 3a. The amount of s-NHSactivated gemcitabine derivative [8] added to the protein solution ofBSA was calculated for a 1:1 molar ratio between the derivative ofgemcitabine [8] and BSA. The mixture of BSA and activated gemcitabinederivative [8] was allowed to stir for 18 hours at room temperature toproduce the conjugate of the activated gemcitabine ester [8] and BSA.This conjugate was then purified by dialysis against 20% DMSO inphosphate buffer (50 mM, pH 7.5) at room temperature. Thereafter theDMSO proportion was reduced stepwise: 10% and 0%. The last dialysis wasperformed against phosphate buffer at 4° C. The purified gemcitabine[9]-BSA conjugate was characterized by UV/VIS spectroscopy.

Example 5b Preparation of BSA Conjugate [9] with Activated HaptenGemcitabine-[13]

A protein solution of BSA was prepared by dissolving 1 g BSA inphosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL.To 10.0 mL of the protein solution of BSA while stirring on ice, wasadded 0.620 mL of s-NHS activated gemcitabine derivative [13] preparedin Example 3b. The amount of s-NHS activated gemcitabine derivative [13]added to the protein solution of BSA was calculated for a 1:1 molarratio between the derivative of gemcitabine [13] and BSA. The mixture ofBSA and activated gemcitabine derivative [13] was allowed to stir for 18hours at room temperature to produce the conjugate of the activatedgemcitabine ester [13] and BSA. This conjugate was then purified bydialysis against 15% DMSO in phosphate buffer (50 mM, pH 7.5) at roomtemperature. Thereafter the DMSO proportion was reduced stepwise: 10%,5%, and 0%. The last dialysis was performed against phosphate buffer at4° C. The purified gemcitabine-BSA conjugate [14] was characterized byUV/VIS spectroscopy.

Example 6 Preparation of Polyclonal Antibodies to Gemcitabine-KLH [9]

Ten female BALB/c mice were immunized i.p. with 100 μg/mouse ofgemcitabine-KLH immunogen [9], as prepared in Example 4, emulsified inComplete Freund's adjuvant. The mice were boosted once, four weeks afterthe initial injection with 100 μg/mouse of the same immunogen emulsifiedin Incomplete Freund's Adjuvant. Twenty days after the boost, testbleeds containing polyclonal antibodies from each mouse were obtained byorbital bleed. The anti-serum from these test bleeds containinggemcitabine antibodies were evaluated in Examples 8 and 9.

Example 7a Microtiter Plate Sensitization Procedure with Gemcitabine-BSAConjugate [9]

The ELISA method for measuring Gemcitabine concentrations was performedin polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with Gemcitabine-BSA conjugate [9] (prepared as inExample 5a) by adding 300 μL of Gemcitabine-BSA conjugate [9] at 10μg/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hoursat room temperature. The wells were washed with 0.05M sodium carbonate,pH 9.6 and then were blocked with 375 μL of 5% sucrose, 0.2% sodiumcaseinate solution for 30 minutes at room temperature. After removal ofthe post-coat solution the plates were dried at 37° C. overnight.

Example 7b Microtiter Plate Sensitization Procedure with Gemcitabine-BSAConjugate [14]

The ELISA method for measuring Gemcitabine concentrations was performedin polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with Gemcitabine-BSA conjugate [14] (prepared as inExample 5b) by adding 300 μL of gemcitabine-BSA conjugate [14] at 10μg/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hoursat room temperature. The wells were washed with 0.05M sodium carbonate,pH 9.6 and then were blocked with 375 μL of 5% sucrose, 0.2% sodiumcaseinate solution for 30 minutes at room temperature. After removal ofthe post-coat solution the plates were dried at 37° C. overnight.

Example 8 Antibody Screening Procedure Titer

This procedure is to find the dilution of antibody to be tested fordisplacement as in Example 9. The ELISA method for screening gemcitabineantibodies (produced in Example 6) was performed with the microtiterplates that were sensitized with gemcitabine-BSA conjugate prepared inExamples 7a and 7b. The antibody screening assay was performed bydiluting the murine serum from test bleeds (as in Example 6) containingpolyclonal gemcitabine antibodies to 1:10, 1:100, 1:1,000 and 1:10,000(volume/volume) in phosphate buffered saline containing 0.1% BSA and0.01% thimerosal. To each well of gemcitabine-BSA sensitized wells(prepared in Examples 7a and 7b) 50 μL phosphate buffered salinecontaining 0.1% BSA and 0.01% thimerosal and 50 μL of diluted antibodywere added and incubated for 10 minutes at room temperature withshaking. During this incubation antibody binds to the gemcitabine-BSAconjugate passively absorbed in the wells (Examples 7a and 7b). Thewells of the plates were washed three times with 0.02 M TRIS, 0.9% NaCl,0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to remove any unboundantibody. To detect the amount of Gemcitabine antibody bound to thegemcitabine-BSA conjugate in the wells, 100 μL of a goat anti-mouseantibody—HRP enzyme conjugate (Jackson Immunoresearch) diluted to aspecific activity (approximately 1/3000) in PBS with 0.1% BSA, 0.05%ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, in this example TMB, were added to each well. After anincubation of 10 minutes at room temperature with shaking, during whichthe goat anti-mouse antibody—HRP enzyme conjugate binds to gemcitabineantibodies in the wells, the plates were again washed three times toremove unbound goat anti-mouse antibody—HRP enzyme conjugate. To developa measurable color in the wells washing was followed by the addition of100 μL of TMB (TMB Substrate, BioFx), the substrate for HRP, to developcolor for 10 minutes shaking at room temperature. Following theincubation for color development, the absorbance was determined at 650nm (Molecular Devices Plate Reader). The amount of antibody in a wellwas proportional to the absorbance measured and was expressed as thedilution (titer) resulting in an absorbance of 1.5. Titers weredetermined by graphing antibody dilution of the antibody measured(x-axis) vs. absorbance 650 nm (y-axis) and interpolating the titer atan absorbance of 1.5. The titer which produced absorbance of 1.5determined the concentration (dilution) of antibody used in the indirectcompetitive microtiter plate assay described in Example 9.

Example 9 Indirect Competitive Microtiter Plate Immunoassay ProcedureDetermining IC₅₀ and Cross-Reactivity for Antibodies to Gemcitabine

The ELISA method for determining IC₅₀ values and cross-reactivity wasperformed with the microtiter plates that were sensitized withgemcitabine-BSA conjugates as described in Examples 7a and 7b. Theanalytes were diluted as follows: gemcitabine was diluted in phosphatebuffered saline containing 0.1% BSA and 0.01% thimerosal over aconcentration range of 0.1 to 500 ng/mL for gemcitabine [9]-BSAmicrotiter plates and gemcitabine [14]-BSA microtiter plates,2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine were diluted inphosphate buffered saline containing 0.1% BSA and 0.01% thimerosal overa concentration range of 0.02 to 0.1 μg/mL for gemcitabine [9]-BSAmicrotiter plates and gemcitabine [14]-BSA microtiter plates. Each ofthe assays were performed by incubating 50 μL of the analyte solutionwith 50 μL of one of the antibodies selected from the polyclonalantibodies produced in Example 6 with the immunogen of Example 4. Theassays were all performed by diluting the concentration of theantibodies in each of the wells to the titer determined in Example 8.During the 10 minute incubation (at room temperature with shaking) thereis a competition of antibody binding for the gemcitabine-BSA conjugatein the well (produced in Examples 7a and 7b) and the analyte insolution. Following this incubation the wells of the plate were washedthree times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001%thimerosal, pH 7.8 to remove any material that was not bound. To detectthe amount of gemcitabine antibody bound to the gemcitabine-BSAconjugate in the wells (produced in Examples 7a and 7b), 100 μL of agoat anti-mouse antibody—HRP enzyme conjugate (Jackson Immunoresearch)diluted to a predetermined specific activity (approximately 1/3000) inPBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal, capable of bindingspecifically with murine immunoglobulins and producing a colored productwhen incubated with a substrate, in this example TMB, were added to eachwell. After an incubation of 10 minutes at room temperature withshaking, during which the goat anti-mouse antibody—HRP enzyme conjugatebinds to gemcitabine antibodies in the wells, the plates were againwashed three times to remove unbound secondary conjugate. To develop ameasurable color in the wells washing was followed by the addition of100 μL of TMB (TMB Substrate, BioFx), the substrate for HRP, to developcolor in a 10 minute incubation with shaking at room temperature.Following the incubation for color development, 50 μL of stop solution(1.5% sodium fluoride in di-H₂O) was added to each well to stop thecolor development and after 20 seconds of shaking the absorbance wasdetermined at 650 nm (Molecular Devices Plate Reader). The amount ofantibody in a well was proportional to the absorbance measured andinversely proportional to the amount of gemcitabine in the sample. TheIC₅₀ values of gemcitabine and 2′,2′-difluoro-2′-deoxyuridine weredetermined by constructing dose-response curves with the absorbance inthe wells plotted versus analyte, concentration in the wells. Theabsorbance of the color in the wells containing analyte was compared tothat with no analyte and a standard curve was generated. The IC₅₀ valuefor a given analyte was defined as the concentration of analyte that wasrequired to have 50% of the absorbance of the wells containing noanalyte. The cross-reactivity was calculated as the ratio of the IC₅₀for gemcitabine to the IC₅₀ value for 2′,2′-difluoro-2′-deoxyuridine andexpressed as a percent. After screening the library of monoclonalantibodies using this method, the monoclonal antibodies were chosen.These chosen antibodies were classified according to their plate andwell number as follows: 5H8-24, 12A5-24, 2F12-24, 14G3-15, 13B12-10,16D6-p-10, and 10G1-11. When measured with these antibodies, the percentcross-reactivities of these antibodies, relative to gemcitabine for2′,2′-difluoro-2′-deoxyuridine (dFdU) were 0.1-0.8%, and the percentcross-reactivities of these antibodies relative to gemcitabine for3,4,5,6-tetrahydrouridine (THU) were 0.0058-0.028%. Results for thegemcitabine monoclonal antibodies are given in the following Tables Iand II.

TABLE I Cross-reactivity of competitive immunoassay using monoclonalantibodies to gemcitabine-BSA [9] (Example 9). Plates coated withgemcitabine-BSA [9] conjugate (Example 9) Gemcitabine dFdU THU % cross-% cross- IC50 IC50 IC50 reactivity reactivity Subclone # (ng/mL) (ng/mL)(ng/mL) dFdU THU 5H8-24 14 2500 >100,000 0.56 <0.014 12A5-24 204900 >100,000 0.41 <0.020 2F12-24 27 4300 >100,000 0.63 <0.027 14G3-1521 3200 >100,000 0.64 <0.021 13B12-10 6 1200 >100,000 0.45 <0.005516D6-p-10 27 5600 >100,000 0.48 <0.027 10G1-11 7 1300 >100,000 0.52<0.0068

TABLE II Cross-reactivity of competitive immunoassay using monoclonalantibodies to gemcitabine-BSA [14] (Example 9). Plates coated withgemcitabine-BSA conjugate [14] (Example 9) Gemcitabine dFdU THU % cross-% cross- IC50 IC50 IC50 reactivity reactivity Subclone # (ng/mL) (ng/mL)(ng/mL) dFdU THU 5H8-24 11 2000 >100,000 0.57 <0.011 12A5-24 54100 >100,000 0.11 <0.0046 2F12-24 28 4200 >100,000 0.68 <0.028 14G3-1514 2300 >100,000 0.61 <0.014 13B12-10 5 800 >100,000 0.64 <0.005116D6-p-10 17 3700 >100,000 0.45 <0.017 10G1-11 8 1300 >100,000 0.61<0.0082

As seen from these tables, the antibodies of this invention aresubstantially selectively reactive with the active form of gemcitabinewith minimal cross-reactivity with both the inactive metabolite2′,2′-difluoro-2′-deoxyuridine and 3,4,5,6-tetrahydrouridine.

1. An immunoassay for detecting gemcitabine in a sample comprisingproviding a mixture of a) said sample, b) an antibody selectivelyreactive with gemcitabine and not substantially cross-reactive with2′,2′-difluoro-2′-deoxyuridine and tetrahydrouridine, and c) a conjugateof a carrier having either a reactive thiol or amino group with acompound of the formula:

wherein B is —CH₂— or

Y is an organic spacing group; X is a functional group capable ofbinding to said carrier through said amino or thiol group; and p is aninteger from 0 to 1 or salts thereof; causing the gemcitibine in thesample and said conjugate in said mixture to bind with said antibody andthereafter measuring the amount of said conjugate in said mixture whichis bound or unbound to said antibody whereby the presence of gemcitibinein the sample can be determined.
 2. The process of claim 1, wherein thesample is a human sample.
 3. The immunoassay of claim 2, wherein saidantibody is generated from an immunogen comprising an immunogeniccarrier having a reactive thiol or amino group conjugated to a compoundof the formula:

wherein p, X, Y and B are as above; or salts thereof. wherein p, X, Yand A are as above.
 4. The immunoassay of claim 3, wherein the carriercontains a thiol group and X is the compound which is linked to theimmunogenic polymer is a functional group capable of reacting with saidthiol.
 5. The immunoassay of claim 4, wherein X is


6. The immunoassay of claim 5, wherein Y is lower alkylene.
 7. Theimmunoassay of claim 6 wherein the immunogenic carrier contains as thefunctional group

wherein v is an integer from 1 to
 6. 8. The immunoassay of claim 2,wherein the antibody is attached to a solid support.
 9. The immunoassayof claim 8, wherein the solid support is microtiter plates.
 10. Theimmunoassay of claim 9, wherein the solid support is nanoparticles. 11.An antibody which binds selectively to gemcitibine and does not have anysubstantially cross reactivity to 2′,2′-difluoro-2′-deoxyuridine andtetrahydrouridine.
 12. The antibody of claim 11 wherein said antibodyhas a cross-reactivity with regard to 2′,2′-difluoro-2′-deoxyuridine andtetrahydrouridine of less than 20%, based upon said antibody'sreactivity with gemcitibine.
 13. The antibody of claim 12 wherein saidcross reactivity is less than 10%
 14. The antibody of claim 11, whereinsaid antibody is derived from mice, sheep, rabbits or rats.
 15. Theantibody of claim 14, wherein said antibody is a monoclonal antibody.16. The antibody of claim 11, wherein said antibody is derived from animmunogenic carrier having a reactive amino or thiol group polymerconjugated to a compound selected from the group consisting of compoundsof the formula:

wherein B is —CH₂— or

Y is an organic spacing group; X is a functional group capable ofbinding to said carrier through said amino or thiol group; and p is aninteger from 0 to 1 or salts thereof:
 17. The antibody of claim 16,wherein the carrier contains a thiol group and X in the compound whichis conjugated to the immunogenic polymer is a functional group capableof reacting with said thiol.
 18. The antibody of claim 17, wherein X insaid compound is


19. The antibody of claim 18, wherein Y in said compound is loweralkylene.
 20. The antibody of claim 19, wherein the immunogenic carriercontains as the functional group:

Wherein v is an integer from 1 to
 6. 21. The antibody of claim 20,wherein said antibody is derived from mice, sheep, rabbits or rats. 22.A compound of the formula:

wherein B is —CH₂— or

Y is an organic spacing group; X is a functional group capable ofbinding to said carrier through said amino or thiol group; and p is aninteger from 0 to 1 X is a functional group capable of binding to athiol or an amino group; and B is —CH₂— or or salts thereof.
 23. Thecompound of claim 22, wherein p is
 0. 24. The compound of claim 21,wherein X is

—N═C═R_(4′)

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 25. The compound ofclaim 24, wherein X is

and R₃ is hydrogen.
 26. The compound of claim 24, wherein X is

and OR₃ forms a reactive ester.
 27. The compound of claim 26, whereinthe ester formed is a lower alkyl ester, imidoester or amidoester. 28.The compound of claim 22, wherein p is
 1. 29. The compound of claim 28,wherein X is

—N═C═R_(4′)

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 30. The compound ofclaim 28, wherein Y is alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 31. A conjugate of a carrier having a thiol or amine group with acompound of the formula:

wherein B is —CH₂— or

Y is an organic spacing group; X is a functional group capable ofbinding to said carrier through said amino or thiol group; and p is aninteger from 0 to 1 or salts thereof:
 32. The conjugate of claim 31,wherein p is
 0. 33. The conjugate of claim 32, wherein p is
 1. 34. Theconjugate of claim 33, wherein Y is alkylene containing from 1 to 10carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 35. The conjugate of claim 34, wherein the carrier contains animmunogenic polymeric polymer containing one or more amino groups linkby

wherein R₄ is oxygen or sulfur.
 36. A kit for determining the presenceof gemcitibine in a patient sample comprising reagents in separatecontainers, one of the reagents being a conjugate of a carriercontaining a functional amino or thiol group with a compound selectedfrom the groups consisting of compounds of the formula:

wherein B is —CH₂— or

Y is an organic spacing group; X is a functional group capable ofbinding to said carrier through said amino or thiol group; and p is aninteger from 0 to 1 or salts thereof; and the second containercontaining an antibody substantially selectively reactive withgemcitibine and not substantially cross-reactive to2′,2′-difluoro-2°-deoxyuridine and tetrahydrouridine
 37. The kit ofclaim 36, wherein said conjugate is present in a predetermined amount insaid first container.
 38. The kit of claim 37, wherein said kit is usedto determine the amount of gemcitibine in said sample.
 39. The kit ofclaim 38, wherein said carrier has a reactive terminal functional thiolgroup and X is a terminal functional group capable of binding to saidthiol group.
 40. The kit of claim 39, wherein X is